Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), the most common human genetic cancer predisposition syndrome. Individuals with NF1 suffer from a wide range of malignant and nonmalignant clinical manifestations including plexiform neurofibromas (PN), complex precancerous lesions which affect 25-40% of NF1 patients and cause major lifelong morbidity and mortality. The NF1 gene encodes neurofibromin, a GTPase-activating protein (GAP) for p21ras (Ras). We previously determined that loss of a single allele of Nf1 (Nf1+/-) results in Ras hyperactivation in NF1 patient (NF1+/-) and murine (Nf1+/-) myeloid cells, a concept known as haploinsufficiency. We generated a genetically engineered murine model of neurofibromatosis and demonstrated that plexiform neurofibroma formation requires the inflammatory contribution of Nf1+/- bone marrow. Further, we showed that genetic inhibition of kinase pathways downstream of neurofibromin in the hematopoietic system prevents tumorigenesis. This work has led to the first ever successful pharmacological treatment of these tumors in both preclinical models and in our phase II clinical trial. Despite this success, complementary strategies to correct Ras hyperactivation in neurofibromin-deficient tissues are needed due to the complexity of Ras-mediated signaling pathways and the heterogeneity of patient response to kinase inhibitors for these complex tumors that are completely resistant to traditional chemotherapy and radiation treatment. Neurofibromin is phosphorylated, ubiquitinated, and degraded at the proteasome in response to growth factor stimulation, but little is known about the mechanistic aspects of this process. Specifically, the ubiquitin ligase specificity factor(s) (E3) that govern neurofibromin degradation in the tumor-driving hematopoietic cells are not known. The F-box ubiquitin ligases degrade selected proteins in a phosphorylation-dependent manner, leading us to hypothesize that the NF1 E3 belongs to the F-box family. In initial unpublished studies pursued in preparation for this application, we conducted an RNAi screen, which identified strong novel candidate F-box ubiquitin ligases for NF1. Here, we propose to mechanistically examine these newly identified F-box proteins modulating neurofibromin degradation ex vivo and in vivo. We also propose to employ an unbiased functional genomics strategy to identify kinase(s) that promote F-box-dependent degradation of neurofibromin via the ubiquitin-proteasome pathway. We will employ a multidisciplinary approach to determine whether disruption of our candidate neurofibromin E3s in vivo can rescue neurofibromin haploinsufficiency and prevent plexiform neurofibroma initiation and progression. These studies will also provide basic insights into the regulation of a common but understudied tumor suppressor gene.